Chemical composition, kinetic study and antimicrobial activity of essential oils from Cymbopogon schoenanthus L. Spreng extracted by conventional and microwave-assisted techniques using cryogenic grinding

Industrial Crops & Products 139 (2019) 111505

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Chemical composition, kinetic study and antimicrobial activity of essential oils from Cymbopogon schoenanthus L. Spreng extracted by conventional and microwave-assisted techniques using cryogenic grinding

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Fatima-Zohra Bellika, Farid Benkaci-Alia, , Zouheir Alsafrab, Gauthier Eppeb, Samira Tatac, Nasserdine Sabaouc, Racim Zidanid a

Laboratory of Functional Organic Analysis, Department of Organic Chemistry, Faculty of Chemistry, University of Sciences and Technology Houari Boumediene (U.S.T.H. B), El Alia, BP 32, Bab Ezzouar, 16111, Algiers, Algeria Laboratoire de Spectrometrie de Masse L.S.M, University of Liege, Allée du 6 Août, Bât B6c, 4000, Liège, Sart-Tilman, Belgium c Department of Biology, Laboratory of Biology of the Microbial Systems, Ecole Normale Supérieure El Bachir El Ibrahimi (E.N.S), Kouba-Alger, BP 92, Algeria d SYMAO, 15 rte de fay 01660 mezeriat, France b

A R T I C LE I N FO

A B S T R A C T

Keywords: Volatile oil Microwave - assisted distillation Cryogenic grinding GC–MS Antimicrobial activity Cymbopogon schoenanthus L. Spreng (Poaceae)

In this present work, volatile oils from Cymbopogon schoenanthus L. Spreng’s (CS) leaves extracted by microwave assisted hydrodistillation (MAHD) and microwave assisted steam distillation (MASD) were studied according to simple (SG) and cryogenic grinding (CG) and compared with conventional hydrodistillation (HD). Extraction time, kinetics, energy consumption, CO2 rejected, physical properties, chemical composition and antimicrobial activity were investigated for the first time with this specie using microwave extraction and showed that MAD is a promising and innovative technique for extraction of volatile oils; also, cryogenic grinding allowed a high extraction yield compared to the classical grinding (MAHD-CG: 1.76% - MAHD-SG: 1.25%) and (MASD-CG: 1.5% - MASD-SG: 1.11%). GC and GC–MS analysis showed variability in composition according to the technique used especially in major constituents and in chemical classes. Qualitative and quantitative kinetic study demonstrated a significative effect of extraction technique and grinding mode on aromatic profile. Evaluation of antimicrobial activity showed that the efficiency of volatiles from CS varied according to extraction technique used where MASD-SG volatile was the most effective one.

1. Introduction Cymbopogon schoenanthus L. Spreng (CS) commonly named as Camel grass or Lemongrass, known in Algeria, as ‘Lemmad’. Is a sub-spontaneous (Naima et al., 2016), perennial (Khadri et al., 2010), aromatic/ medicinal plant that belongs to the Poaceae family (Cafferty et al., 2000). Cymbopogon genus includes 56 species widely spread through the Mediterranean area (Lino et al., 2010). Native to tropical Asia, this plant is now distributed in Asia and in north and central Africa (Alsnafi, 2016). Growing in dry stony places (Hashim et al., 2016). Cymbopogon schoenanthus L. Spreng was traditionally used to treat kidney and urinary diseases, digestive diseases, rheumatisms, fever, food poisoning (Ramdane et al., 2015), intestinal troubles and for bringing back the appetite (Khadri et al., 2008). In Menia (Algeria) where the plant was collected for this experimental study, herb decoction is used as

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mouthwash to relief dental pain, and to diminish cramping pain for pregnant women before delivery. Essential oil isolated from this plant is used in perfumery, cosmetics and pharmaceutics (Khadri et al., 2010).Volatile oils can be isolated from aromatic plants using several techniques including hydrodistillation (HD) which has been the traditional technique for essential oil extraction. However, this method is high consuming time (causing thermal degradation) and energy as well as releasing considerably carbon dioxide in the atmosphere. While, many other techniques based on microwave heating constitutes a good alternative for conventional processes (Benkaci-Ali et al., 2007), because of their low extraction time and energy consumption. Hence, the extraction cost without adverse effecting essential oil quality as well as enhancing extraction efficiency (Benkaci-Ali et al., 2006; Mandal et al., 2007). In recent years, microwave assisted extraction (MAE) has proven it

Corresponding author. E-mail address: [email protected] (F. Benkaci-Ali).

https://doi.org/10.1016/j.indcrop.2019.111505 Received 7 February 2019; Received in revised form 16 June 2019; Accepted 22 June 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

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efficiency for the extraction compared to traditional techniques being time and energy consuming, generating thermal and hydrolytique degradations of thermolabile components, devaluing the quality of oils. Besides, grinding process which precedes extraction is performed in order to facilitate liberation of aroma by reducing size of particle and increasing plant material surface (Murthy et al., 1999). However, this procedure can cause loss of volatiles (Fornari et al., 2012), in addition to thermal degradation for thermolabile compounds due to heat generation that accompanies frictional forces (Kaur and Srivastav, 2018). Cryogenic grinding or cryo-grinding (CG) uses cryogenic liquids in order to reduce temperature between the two rubbing surfaces preventing the easily oxidative compounds from degradation which preserves the quality of plant material. Liquid nitrogen (N2) supplies refrigeration effect at −196 °C and has been used in many studies in agricultural field (Mékaoui et al., 2016; Singh and Goswami, 2000). To the best of author’s knowledge, no work has been published on kinetic extraction, characterization or evaluation of biological activity of essential oil isolated from cryogrinded Cymbopogon schoenanthus L. Spreng. Therefore, the objectives of this present work is to investigate the chemical composition, extraction kinetics and antimicrobial activity of Cymbopogon schoenanthus L. Spreng essential oil using microwave assisted hydrodistillation (MAHD) and microwave assisted steam distillation (MASD) performing two grinding modes as classical and cryogenic grinding (N2 at −196 °C) in terms of comparison. A qualitative and quantitative comparison was made with conventional hydrodistillation. Our objective is to make a convenient comparison in terms of extraction time, energy consumption, environmental impact, yield, physical properties, chemical composition and antimicrobial activity.

2.4. Extraction procedure

2. Experimental

2.4.3. Microwave assisted Steam distillation (MASD) apparatus and procedure 40 g of grinded leaves were submitted to MASD using a simple column (Height =40 cm, Diameter =3 cm) coupled to a Clevenger-type apparatus and treated with 300 ml of water. The power used was 600 W in order to avoid the hazards of overpressure in the extraction apparatus. The plant was exposed to steam without been immersed into water. After condensation, the collected essential oil was dried under anhydrous sulphate and stored at a temperature of 4 °C in a sealed amber vial until used for analysis. Each extraction was carried out in triplicate.

Extraction of the essential oils from CS leaves was performed using five different methods HD, MAHD-SG, MAHD-CG, MASD-SG and MASD-CG. Extraction temperature is equal to water boiling temperature at atmospheric pressure (100 °C). 2.4.1. Hydrodistillation (HD) apparatus and procedure 100 g of grinded CS leaves were submitted to conventional hydrodistillation using a Clevenger-type apparatus (Pharmacopée Européenne, 1996), immersed with 1 L of distilled water. The collected essential oil was dried under anhydrous sulphate and conserved at 4 °C in a sealed amber vial until used for analyses. Each extraction was realized three times. 2.4.2. Microwave assisted hydrodistillation (MAHD) apparatus and procedure A mass of 50 g of grinded CS leaves were soaked with 375 ml of solvent (distilled water) in a 1 liter capacity round flask. The MAHD has been performed using microwave laboratory as described in reference (Mékaoui et al., 2016) at atmospheric pressure using a power of 800 W. Ratio (mass of plant material /volume of water) and microwave power were optimized in a preliminary study. In fact, four microwave powers were tested: 500 W, 600 W, 800 W and 1000 W. It was shown that for MAHD, increasing the power accelerated the time of extraction and allowed to raise the yield of essential oil, but at 1000 W, a light smell of burning has been noted; thus, the power value was fixed at 800 W. The volatile oil isolated was dried under anhydrous sulphate and preserved at 4 °C in a sealed amber vial until used for analysis. Each extraction was achieved three times.

2.1. Plant material Cymbopogon schoenanthus L. Spreng (CS) was collected from Oued Sbaa, El-Menia, ALGERIA (Latitude: 30° 48′ N; Longitude: 2°50′ E; Altitude 420 m) in March 2017. The Identification of the investigated specie was confirmed in the herbarium of Botany Department, National superior School of Agronomy (ENSA), Algiers. The plant material was air-dried in the shade, sifted to remove sand residues, then, leaves were separated from roots using pruning shears, leaves were stored at room temperature in a moisture-free atmosphere until extraction. The mass of CS’s leaves were 100, 50 and 40 g for HD, MAHD and MASD respectively.

2.4.4. Extraction kinetics For the kinetic experiments, the same conditions were applied for all extraction methods; each sample of volatile oil was collected at different times (the sampling times differed from method to another). Each fraction was weighted to measure the yield, and stored at 4 °C until used for chromatographic analyses. The operation was carried out in duplicate.

2.2. Chemicals Alkane standard solutions C6-C28 (40 mg/ml) and the internal standard C12, were purchased from Sigma-Aldrich (Steinheim, Germany), Mueller-Hinton agar (MHA) and Sabouraud dextrose agar (SDA) were bought from Merck (Darmstadt, Germany), Dimethyl sulfoxide (DMSO) was purchased from Carlo Erba (Val de Reuil, France). All other solvents were of analytical grade purchased from Fluka and Sigma-Aldrich.

2.5. Physical constants Specific gravity and refractive index of essential oils from Cymbopogon schoenanthus L. Spreng extracted by the five methods have been determined as reported by standard methods (Agence française de Normalisation, 2007): specific gravity corresponds to the ratio of the weight of the essential oil to the weight of an equal volume of water at the same temperature. Refractive index was measured using an ATAGO refractometer at 20° ± 1 °C.

2.3. Simple and cryogenic grinding Rotor mill (Model Pulverisette 14, Fritsch, Germany) was worn for simple (SG) and cryogenic grinding (CG) with a peripheral rotors speed of 70 (15,000 RPM) m/ second. Plant material was grinded to high particle size particle size (dp1) and low particle size (dp2) for SG and CG respectively. The cryo-grinding was performed by pouring liquid nitrogen at −196 °C to the vegetable matrix.

2.6. GC-FID analysis Gas chromatography analysis was performed using a 6890 series instrument (Agilent Technologies, Palo Alto, CA, USA) gas chromatograph equipped with flame ionization detector (FID), the conditions 2

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during 24 h for bacterial strains and at 30 °C during 48 h for fungal strains. Antimicrobial activity was estimated by measuring diameter of inhibition zones (mm). Amoxicillin was used as positive control. The antimicrobial activity is expressed as the zone of inhibition (in millimeters) surrounding the discs (including diameter of disc). Antimicrobial activity potential is estimated according the size of inhibition zone, it is considered to be great (inhibition zone ≥ 20 mm), moderate (inhibition zone 15–19 mm), weak to moderate (inhibition zone 9–15 mm) or weak (inhibition zone < 9 mm). All essays were performed three times.

followed were: fused-silica capillary column HP5-MS (60 m, 0.25 mm i.d, 0.25 μm film thickness, 5% biphenyl, 95% dimethyl polysiloxane). Carrier gas Helium (0.03 MPa, flow rate 0.5 ml/min), injector and detector temperature were regulated at 280 and 300 °C. 1 μl of each sample (oil diluted in hexane at 1%) was injected in the splitless mode, oven temperature progress was from 60 °C for 10 min, then increased from 60 to 250 °C at 4 °C/min and held at 250 °C for 10 min. 2.7. GC–MS analysis Gas Chromatography-mass spectrometry (GC–MS) analysis was achieved using same gas chromatograph and column (HP5-MS capillary column, 60 m, 0.25 mm i.d, 0.25 μm film thickness) coupled to a MSD 5973 mass spectrometer. Oven temperature was programmed at 60 °C for 8 min, then, increased from 60 to 250 °C at 4 °C/min and held at 250 °C during 10 min. in a splitless mode and injection volume of 1 μl (samples were diluted in hexane at 1%). Helium was used as carrier gas (flow rate of 1.2 ml/min). Injector and detector temperatures were kept at 250 and 280 °C respectively. Scan time: 1.5 s, splitless time 0.75 min. For MS detection, The MS source temperature was 230 °C; MS quadrapole temperature at 150 °C; mass scan, 30–550 amu and interface temperature at 290 °C. ChemStation software was used to handle chromatograms and mass spectra. The volatile components were identified comparing their GC retention indices (RI) on a HP5-MS column calculated according to Van den Dool and Kratz (Van Den Dool and Dec. Kratz, 1963) determined with reference to an homologous series of C6-C28 n-alkanes with those of authentic standards (authors laboratory), and by comparing their mass spectral fragmentation patterns with literature (Adams, 2007) and wiley / NBS library as data bank. Quantification was done using retention factors relative to dodecane (internal standard). Percentages of components were calculated as average values of three analyses.

2.8.3. Determination of minimum inhibitory concentration (MIC) Minimum inhibitory concentrations of different essential oils were performed using broth micro-dilution assay according to National Committee for Clinical Standards Guidelines (Jorgensen, 1993) with slight modifications. For each sample of tested essential oils, an initial solution of 1500 μg/ml was prepared in Dimethyl Sulfoxide (DMSO), then, serial dilutions were realized with the same solvent to obtain concentrations from 1 to 1500 μg/ml. Afterwards, each dilution was added to Agar medium 10:90 (v/v) (MHA for bacterial strains and SDA for fungal strains) in order to obtain concentrations from 0.1 to 150 μg/ ml. those resulted mixtures were immediately poured into Petri dishes. Fresh cultures of each strain tested in agar disc diffusion method (1 × 106 CFU/ ml for bacterial strains and 1 × 106 spore/ ml for fungal strains) were prepared and inoculated in those Petri dishes, then, incubated at 30 °C during 24 h for bacterial strains and at 30 °C during 48 h for fungal strains. Standard antibiotic Amoxicillin was tested for comparison using the same concentrations. DMSO was used as negative control. MIC corresponds to the lowest concentration that inhibits growth of microbial strains. All experiments were performed in triplicate. 2.9. Statistical analysis

2.8. Antimicrobial activity

Experimental results were recorded as means ± standard deviation of three values (n = 3) using Microsoft Excel statistical analysis program. K-Means clustering and Principal Component Analysis (PCA) were performed by R programming language using RStudio software (RStudio, Inc., Boston, Massachusetts, USA).

Antimicrobial activity of Cymbopogon schoenanthus L. Spreng essential oils isolated by the five methods was evaluated using two tests: Agar disk diffusion method and determination of Minimum Inhibitory Concentration (MIC). 2.8.1. Microbial strains Antimicrobial activity of CS essential oils was evaluated against two Gram-positive bacteria: Staphylococcus aureus MRSA 639cand Listeria monocytogenes ATCC 13932, three Gram-negative bacteria: Klebsiella pneumoniae E40, Salmonella Typhi ATCC 14028 and Escherichia coli ATCC 8739, three fungi: Aspergillus westerdijkiae ATCC 3174, Fusarium culmorum ATCC 36017 and Aspergillus flavus NRRL 3251) and one yeast: Candida albicansM3. Regarding experiments, microorganisms were cultured in MuellerHinton Agar broth (MHA) for bacteria (at 30 °C for 24 h) and Sabouraud dextrose Agar broth (SDA) for filamentous fungi (at 30 °C for 48 h).

3. Results and discussion 3.1. Effect of extraction technique and time on yield of essential oil To assess the influence of extraction time on the essential oil yield, the overall extraction curves were constructed using the cumulated mass of volatile oil collected at different time intervals. Variation of extraction yield (%) according to time (min) for the different methods used is represented by (Fig. 1) observing three phases. The first step is

2.8.2. Antimicrobial activity by Agar disc diffusion method Paper disk diffusion method was used for the determination of antimicrobial activity as described by Djouahri et al. (2017) with slight modifications: microbial suspensions were prepared by mixing and vortexing fresh active cultures with physiological water; optical densities were adjusted to 1 × 106 CFU/ml for bacterial strains and to 1 × 106 spore/ml for fungal strains. Then, spread using a sterile cotton swab in post-autoclaving MHA medium for bacterial strains and SDA medium for fungal strains (100 μl suspension /100 ml medium) in 90 mm Φ Petri dishes. 6 mm Φ filter paper discs were sterilized under UV hood for 45 min, afterward, individually impregnated with 10 μl of essential oil, then, placed at the surface of agar Petri dishes using sterilized tweezers. Subsequently, these Petri dishes were kept at 4 °C for 1 h for essential oil diffusion in the medium, then, incubated at 30 °C

Fig. 1. Variation of global (accumulated) yield (%) according to the extraction time (min) for the different methods. 3

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an increasing line AB’, and the curvilinear B’B defining the first quantities of volatile oil located in exogenous sites of the plant, this last is marked by a decline in extraction yield which diminish gradually until reaching the point B. During the first AB step the amount of EO extracted by the different techniques HD, MAHD-SG, MAHD-CG, MASDSG and MASD-CG are respectively 53.25, 62.07, 39, 92.4 and 85.74%. The second step is represented by another curvilinear branch BC characterizing the penetration of solvent (water) into the endogenous sites of the plant causing the diffusion of essential oil from the midst of particles via external environment. This stage took from 20 to 210 min depending on the technique used and over of 75% of the remaining amount of volatile oil has been extracted. The third phase consists of a horizontal line marking the end of extraction. Speed of extraction differs from a technique to another noting that MASD is most rapid process than MAHD (40–50 min compared to 120–150 min respectively) and HD (240 min), which leads to conclude that microwave hydrodistillation (MAHD) is better than conventional hydrodistillation, in terms of short extraction time, cleanliness (using water as a green and an alternative solvent for extraction) (Filly et al., 2016) and energy saving as confirmed by many studies (Akloul et al., 2014; Benkaci-Ali et al., 2007; Bouchachia et al., 2017; Chemat et al., 2006). Table 1 shows variation of essential oil’s yield according to extraction technique and time. The HD technique provided, at 180 min of extraction, a yield of 1.92 ± 0.01% while MAHD-SG at 45 min and MASD-SG at 40 min allowed extracting 1.25 ± 0.14 and 1.11 ± 0.07% respectively. We point out that the total extraction time was chosen to be lower than the one recorded in the kinetic study, in order to prevent any thermal degradation reactions of thermolabile compounds and allowing accurate evaluation of the chemical composition of the volatile oils isolated. Cryogenic grinding process increased the recovered EO’s yields in the same extraction time MAHD-CG at 45 min (1.76 ± 0.09%) and MASD-CG at 40 min (1.5 ± 0.03%) compared to simple grinding techniques. The cryogenic grinding (N2 at −196 °C) helps to maintain low temperature by absorbing the generated heat during grinding process (Akloul et al., 2014), this heat represents almost 99% of the generated energy, whereas only 1% is used for real grinding (Tischer et al., 2017). In addition we noted a significative variation of essential oil yield according the grinding mode and particle size (PS). The CG essential oil recovery (low particle size) was higher than those of classical or simple grinding (high particle size). This may be explained by the fact that when the particles become finer, the solid-liquid contact surface of the plant material increases, allowing a better solvent permeation and improved extraction. In addition to keeping the temperature lower and

reducing the size of particles, the vapors from the liquid nitrogen create an inert and dry environment giving supplementary protection of plant quality (Saxena et al., 2018). In agreement with our study, authors (Sharma et al., 2016) showed that cryogenic grinding technology had significantly raised the amount of extracted essential oil with high quality. Extraction times and stages (kinetic) of highlighted techniques are listed in Table 1. For economical (time and energy saving) and ecological (reducing the quantity of CO2 released in the atmosphere) purposes, it is better to choose an optimal extraction time permitting the extraction of 80% of total yield, and according to that, the chosen times for highlighted techniques are approximately: 120 min (HD), 50 min (MAHD-SG), 30 min (MAHD-CG), 15 min (MASD-SG) and 15 min (MASD-CG). In addition, it is preferable to limit the time at these values for the reason of preventing the thermal degradation reactions (hydrolytic and oxidative) of thermolabile compounds. In the whole, performing CG as well as increasing the applied energy (600 and 800 W) accelerated extraction time, but raising the power can cause thermal and hydrolytic degradations. Indeed, when we attempted to investigate the extraction process at 1000 W, a light smell of burning has been noticed; as a consequence, we avoided working above 800 W.

3.2. Cost, energy consumption and environmental impact In term of extraction cost, MASD technique showed a clear advantage over MAHD and conventional HD. Regarding the energy consumption, HD was the most high consuming energy technique (1.548 KWh) recorded in our study. In addition, MASD-SG (0.510 KWh) and MASD-CG (0.517 KWh) techniques were low consuming energy compared the MAHD-SG (0.854 KWh) and MAHD-CG (0.868 KWh). As shown in Table 1, HD required before extraction a heating time of 26.58 min raising the temperature of water (1000 ml) and the plant material (100 g) up to 100 °C, while MAHD took 19–20.30 min, however MASD needed only 11.92–12.53 minutes with a slight augmentation when the extraction is performed by cryogenic grinding. The last, needs more time to heat the frozen plant material treated by liquid nitrogen. From where, microwave assisted techniques reduces significatively energy consumption compared to conventional hydrodistillation as also confirmed by many authors (Asghari et al., 2012; Bouchachia et al., 2017; Ferhat et al., 2007). For environmental impact, the carbon dioxide released in the atmosphere was calculated according to the fact that 1 KWh generated by the combustion of coal or other fossil fuel, releases 800 g of CO2 into the atmosphere (Bernard, 2001). The higher amount of CO2 rejected were recorded in HD technique (1238 g) followed by MAHD (SG: 683 – CG: 694.14 g) and MASD (SG: 408.24 – CG: 413.34 g).

Table 1 Effect of different parameters and extraction technique on yield, energy consumption, CO2 released in the atmosphere and extraction stages of CS essential oil recorded by HD, MAHD-SG, MAHD-CG, MASD-SG and MASD-CG.

Mass of plant material (g) Volume of water (ml) Heating time (min) Extraction time after heating (min) Total extraction time (min) Yield (% w/w) Electric consumption (kWh) CO2 released (g) Extraction steps AB' (min) B'B (min) BC (min) CD (min)

HD

MAHD-SG

MAHD-CG

MASD-SG

MASD-CG

100 1000 26.58 180 206.58 1.92 ± 0.01 1.548 1238.016

100 750 19 45 64 1.25 ± 0.14 0.854 683

100 750 20.13 45 65.13 1.76 ± 0.09 0.868 694.144

40 300 11.92 40 51.92 1.11 ± 0.07 0.510 408.24

40 300 12.53 40 52.53 1.5 ± 0.03 0.517 413.336

10 20 210 30

20 10 120 50

2 3 115 60

5 15 20 10

5 15 30 10

AB: first step of extraction (AB’: linear profile representing the extraction of the essential oil situated in the external solid surface, B’B: curvilinear, characterized by a reduction in extraction rate); BC: second step of extraction; CD: third step of extraction. 4

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complex mixture of secondary metabolites and rich in oxygenated sesquiterpenes, generally, the oxygenated compounds are strongly fragrant, making them much valuable (Akloul et al., 2014). Additionally, it is important to note that extraction technique has significant effect on qualitative and quantitative composition of CS’s essential oil. Indeed, Table 3 reveals that oxygenated sesquiterpenes were present in larger amount in HD essential oil (50.37%) compared to microwave techniques MAHD and MASD. This is may be explained by the fact that HD, long heating time based, allowed the penetration of water (polar solvent) into the internal sites of the plant extracting a greater amount of oxygenated compounds with higher molecular weight. However this technique gives a low fraction of oxygenated monoterpenes (8.47%) and monoterpene hydrocarbons (14.9%). On other hand, using cryogenic grinding permitted rapid extraction with considerable monoterpene hydrocarbons yields (MAHD-CG: 20.86%, MAHD-SG: 20.2% and MASD-CG: 26.66%, MASD-SG: 14.96%) due to the loss of this compounds during the classical grinding because of their lower boiling point (Tischer et al., 2017). Hence, liquid nitrogen at −196 °C allowed decreasing the loss in monoterpene volatiles by cooling the plant material during the grinding as well as enhancing the extraction of monoterpenes compared to conventional hydrodistillation by reducing the time of heating processes causing their thermal degradation. A non negligible compounds (in traces) were also isolated only specifically according the technique achieved such as verbenene (MASD-CG), neoiso-dihydro carveol acetate (MASD-SG), guaiol (MASDSG), aristolone (HD), 14-hydroxy-δ-cadinene (HD) and cryptomeridiol (HD and MASD-SG). Besides, traces of three diterpenes (carissone, Z,Egeranyl linalool and manool) were exclusively extracted by HD. In addition, seven other components were totally absent in the EOs as Z-βocimene (MASD-SG), trans-carveol (HD and MASD-CG), cis-thujopsadiene (MASD-SG and MASD-CG), nootkatene (MAHD-CG), eudesma4(15),7-dien-1β-ol (MASD-CG), iso-longifololacetate (MASD-SG) and totarene (MASD-CG). In agreement with previous works (Bossou et al., 2015; Hashim et al., 2016; Yagi et al., 2016; Yentema et al., 2007), among the most representative components of Cymbopogon schoenanthus L. Spreng oil were eudesmane derivatives. In our study, seven eudesmane-type sesquiterpens were identified representing from 14.1 to 25.3% of total chemical composition : 5-epi-7-epi-α-eudesmol (1.11–2.16%), γ-eudesmol (0.1–2.44%), α-eudesmol (11.64–17.89%), 7-epi-α-eudesmol (0.61–1.2%), eudesma-4(15),7-dien-1β-ol (0.03–0.07%), eudesm7(11)-en-4-ol (0.61–2.34%) and eudesm-7(11)-en-4-ol, acetate (0.02–0.05%). Eudesmols belong to sesquiterpenoid alcohols having several pharmacological effects (Britto et al., 2012). Previous studies realized by Britto et al. (2012) showed that antitumor effect of essential oil from Guatteria friesiana is related to its major constituents (α-, β-, and γeudesmol). Other research showed that eudesmols possess a strong antifungal activity (Mori et al., 2000; SeonHong et al., 2016).

Table 2 Physical properties of Cymbopogon schoenanthus L.Spreng essential oils isolated by different extraction techniques.

Refractive index at 20 °C Relative density at 20 °C

HD

MAHD-SG

MAHD-CG

MASD-SG

MASD-CG

1.4950

1.4900

1.4910

1.4885

1.4890

0.8890

0.8780

0.8730

0.8700

0.8690

Indeed, microwave can be selected as an alternative for the reason of protecting thermolabile compounds, diminishing the amount of CO2 rejected in addition to energy and time economization (Asghari et al., 2012; Bouchachia et al., 2017; Ferhat et al., 2007). 3.3. Physical constants Evaluation of physical properties (refractive index and specific gravity) of essential oil of Cymbopogon schoenanthus L. Spreng extracted by HD, MAHD-SG, MAHD-CG, MASD-SG and MASD-CG are displayed in Table 2. Both properties fall within the ranges 1.478–1.500 (at 20 °C) for refractive index and 0.869- 0.904 (at 20 °C) for specific gravity (Alsnafi, 2016) with a slight difference noted between oils extracted by the microwave techniques (MAHD and MASD) compared to HD volatile oil, due to their chemical composition changing depending on the technique used. 3.4. Effect of extraction technique on chemical composition Chemical composition of Cymbopogon schoenanthus L. Spreng essential oils isolated using the different methods HD, MAHD-SG, MAHDCG, MASD-SG and MASD-CG are displayed in Table 3. In total, 109 components were identified in the volatile oils isolated by all methods studied constituting 99.33% to 99.73% of the essential oil isolated. The major component for all techniques studied was α-eudesmol at different concentration (HD: 17.89%; MAHD-SG: 14.18%; MAHD-CG: 15.54%; MASD-SG: 15.91% and MASD-CG: 11.51%). Based on previous works, it was mentioned that α-eudesmol has antimicrobial activity (Costa et al., 2008), antitrypanosomal activity (Otoguro et al., 2012) and useful for the treatment of neurogenic inflammation and brain injury (Asakura et al., 2000), this compound was also identified in the CS essential oil of Saudi Arabia (11.5%) (Hashim et al., 2016), Benin (2.1%–3.5%) (Bossou et al., 2015), Tunisia (0.4%–5.1%) (Khadri et al., 2008) and Illizi from Algeria (1.8%) (Naima et al., 2016) with piperitone as major constituent, except from Tunisia CS with limonene as major compound. This shows clearly the variability of the chemical composition of Cymbopogon schoenanthus L. Spreng’s essential oil related to geographical factors. Other major components were also identified (Fig. 2) such as transdauca-4(11),7-diene (10.39–12.58%); α-muurolol (3,77–6,55%); hinesol (3.72–6.1%); allo-ocimene (5.02–7.71%) and δ-2-carene (2.78–5.19%). Thus, as observed in Table 3, α-eudesmol and hinesol (oxygenated sesquiterpenes) were present in high amount in the HD essential compared to allo-ocimene and isopulegol which were better extracted from microwave techniques. Regarding, δ-2-carene and sylvestrene (monoterpene hydrocarbons) the highest percentages were recorded with MASD-CG compared to trans-dauca-4 (11),7-diene with MASD-SG. As shown in Table 3, we noted a predominance of oxygenated sesquiterpenes (30.28–50.37%) followed by sesquiterpenes hydrocarbons (23.49–29.63%), monoterpene hydrocarbons (14.91–26.66%) and oxygenated monoterpenes (8.47–13.62%) as well as traces of diterpenes (totarene, carissone, Z,E-geranyl linalool and manool). This confirms that essential oil from Cymbopogon schoenanthus L. Spreng is a

3.5. Effect of extraction technique on chemical variability of essential oil (statistical analysis) In order to better evaluate the chemical variability of Cymbopogon schoenanthus L. Spreng’s essential oils according to extraction technique, data analysis with K-means clustering and Principal Component Analysis (PCA) based on a scaled and centered matrix linking percentages of components to extraction techniques (HD, MAHD-SG, MAHDCG, MASD-SG and MASD-CG) were performed using R programming language. Clustering classification permitted to distinguish three groups of essential oils, the group 1 including HD and MASD-SG essential oils; Class 2 combining MAHD-SG with MAHD-CG essential oils and class 3 corresponding to MASD-CG essential oil. This shows that composition is much related to the technique and grinding mode used, from where a 5

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Table 3 Chemical composition of Cymbopogon schoenanthus L. Spreng extracted by the different Methods. N°

compound

RI

RI (lit.)

HD

MAHD-SG

MAHD-CG

MASD-SG

MASD-CG

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74

Verbenene Cis-meta-mentha-2,8-diene Dehydro-1,8-Cineole δ-2- Carene δ-3-Carene α-Terpinene p-Cymene Sylvestrene Z- β-Ocimene y-Terpinene Terpinolene p-Cymenene Exo-Fenchol Allo-ocimene Cis-Verbenol Isopulegol p-Mentha-1,5-dien-8-ol Terpinen-4-ol Cryptone Cis - Piperitol p-Cymen-9-ol Trans- Piperitol Trans-Carveol Neo iso-dihydro carveol Cumin aldehyde Car-3-en-2-one Piperitone p-Menth-1-en-7-al 3'-methoxy-Acetophenone p-Menth-1-en-9-ol δ-Elemene α-Cubebene Neo iso dihydro carveol acetate α-Ylangene α-Copaene Modheph-2-ene β-Bourborene β- Elemene β-Longipinene β-Cedrene β-Duprezianene Cis-Thujopsene β-Gurjunene α-Guaiene 6,9-Guaiadiene α-Himachalene α-neo-Clovene α-Humulene Allo-Aromadendrene Cis-Muurola-4(14),5-diene Cis-Thujopsadiene y-Muurolene Germacrene D Aristolochene Epi-Cubebol α-Muurolene α-Bulnesene Modhephene-8-β-ol Nootkatene 7-epi-α-Selinene δ- Cadinene (E)-iso-y-Bisabolene α-Cadinene Selina-3,7(11)-diene α-Calacorene Trans- dauca-4(11),7-diene β-Calacorene 1α,10α-epoxy-Amorph-4-ene Germacrene D-4-ol Caryophyllene oxide Cis-dihydro-Mayurone Guaiol 5-epi-7-epi-α -Eudesmol 1,10-di-epi-Cubenol

963 985 990 1003 1009 1017 1023 1028 1035 1056 1087 1091 1120 1130 1140 1147 1167 1177 1185 1197 1206 1210 1217 1229 1240 1247 1251 1275 1296 1396 1337 1347 1358 1373 1377 1385 1387 1391 1399 1421 1424 1431 1433 1439 1444 1450 1454 1454 1460 1466 1468 1479 1484 1489 1496 1502 1507 1515 1515 1520 1524 1530 1537 1546 1547 1558 1566 1572 1576 1585 1597 1602 1610 1620

961 983 988 1001 1008 1014 1020 1025 1032 1054 1085 1089 1118 1128 1137 1145 1166 1174 1183 1195 1204 1207 1215 1226 1238 1244 1249 1273 1298 1294 1335 1345 1356 1373 1374 1382 1387 1389 1400 1419 1421 1429 1431 1437 1442 1449 1452 1452 1458 1465 1465 1478 1484 1487 1493 1500 1509 1513 1517 1520 1522 1528 1537 1545 1544 1556 1564 1570 1574 1582 1595 1600 1607 1618

– 0.3 ± 0.02 0.02 ± 0 4.16 ± 0.44 0.57 ± 0.03 0.27 ± 0.03 1.1 ± 0.09 3.38 ± 0.16 0.02 ± 0 0.02 ± 0 0.03 ± 0 0.04 ± 0 0.06 ± 0 5.02 ± 0.34 0.05 ± 0.01 3.67 ± 0.22 0.08 ± 0.02 0.02 ± 0 0.1 ± 0.02 2.07 ± 0.15 0.03 ± 0 1.97 ± 0.09 – 0.02 ± 0 0.03 ± 0 0.08 ± 0.01 0.22 ± 0.06 0.03 ± 0 0.05 ± 0.01 0.02 ± 0 0.09 ± 0.02 0.08 ± 0.02 – 0.08 ± 0.01 0.12 ± 0.09 0.09 ± 0.01 0.02 ± 0 2.56 ± 0.17 0.04 ± 0 0.35 ± 0.07 0.16 ± 0.02 0.27 ± 0.03 0.34 ± 0.04 0.14 ± 0.01 0.06 ± 0 0.06 ± 0 0.12 ± 0.01 0.09 ± 0.01 0.05 ± 0 0.08 ± 0 0.12 ± 0 0.71 ± 0.05 0.21 ± 0.02 0.54 ± 0.01 3.02 ± 0.25 1.9 ± 0.11 0.2 ± 0.04 0.1 ± 0.02 0.07 ± 0 1.75 ± 0.19 2.4 ± 0.17 0.35 ± 0.03 0.79 ± 0.04 0.29 ± 0.04 0.25 ± 0.04 10.67 ± 0.51 0.22 ± 0.01 0.03 ± 0 0.16 ± 0.02 0.45 ± 0.04 0.18 ± 0 – 2.16 ± 0.12 0.16 ± 0.01

– 0.3 ± 0.02 0.02 ± 0 4.32 ± 0.39 1.05 ± 0.05 0.56 ± 0.05 1.35 ± 0.18 4.69 ± 0.19 0.04 ± 0 0.05 ± 0 0.07 ± 0.01 0.06 ± 0 0.02 ± 0 7.71 ± 0.64 0.05 ± 0 5.58 ± 0.31 0.13 ± 0.03 0.03 ± 0 0.13 ± 0.03 3.79 ± 0.28 0.04 ± 0 3.13 ± 0.23 0.03 ± 0 0.04 ± 0 0.05 ± 0 0.18 ± 0.03 0.35 ± 0.05 0.03 ± 0 0.04 ± 0 0.02 ± 0 0.07 ± 0 0.09 ± 0.01 – 0.04 ± 0 0.15 ± 0.07 0.08 ± 0 0.02 ± 0 2.38 ± 0.22 0.03 ± 0 0.31 ± 0.05 0.15 ± 0.02 0.16 ± 0.02 0.38 ± 0.04 0.08 ± 0 0.04 ± 0 0.07 ± 0.01 0.12 ± 0.02 0.11 ± 0 0.04 ± 0 0.06 ± 0 0.11 ± 0.03 0.71 ± 0.03 0.1 ± 0 0.65 ± 0.02 2.65 ± 0.13 1.97 ± 0.1 0.11 ± 0.01 0.04 ± 0 0.02 ± 0 1.64 ± 0.1 2.98 ± 0.17 0.3 ± 0.01 0.91 ± 0.02 0.29 ± 0.02 0.15 ± 0 11.62 ± 0.3 0.18 ± 0.02 0.04 ± 0 0.34 ± 0.03 0.48 ± 0.02 0.17 ± 0.02 – 1.8 ± 0.21 0.16 ± 0.01

– 0.36 ± 0.03 0.03 ± 0 5.19 ± 0.47 0.94 ± 0.06 0.48 ± 0.04 1.16 ± 0.11 4.83 ± 0.2 0.14 ± 0.02 0.05 ± 0 0.06 ± 0 0.05 ± 0 0.07 ± 0.01 7.6 ± 0.47 0.07 ± 0.01 5.22 ± 0.37 0.12 ± 0.09 0.02 ± 0 0.1 ± 0.01 3.51 ± 0.17 0.03 ± 0 2.78 ± 0.17 0.02 ± 0 0.03 ± 0 0.04 ± 0 0.12 ± 0.05 0.31 ± 0.02 0.02 ± 0 0.03 ± 0 0.03 ± 0 0.09 ± 0.01 0.06 ± 0 – 0.06 ± 0.01 0.09 ± 0.02 0.07 ± 0 0.02 ± 0 2.14 ± 0.12 0.03 ± 0 0.35 ± 0.03 0.11 ± 0.01 0.29 ± 0.03 0.35 ± 0.02 0.11 ± 0 0.05 ± 0 0.07 ± 0 0.11 ± 0 0.09 ± 0 0.04 ± 0 0.08 ± 0.01 0.02 ± 0 0.15 ± 0.02 0.47 ± 0.02 0.83 ± 0.04 1.96 ± 0.11 1.52 ± 0.09 0.15 ± 0.03 0.05 ± 0.01 – 1.29 ± 0.09 2.11 ± 0.09 0.3 ± 0.02 0.67 ± 0.03 0.21 ± 0 0.14 ± 0.01 11.1 ± 0.46 0.25 ± 0.01 0.03 ± 0 0.36 ± 0.03 0.4 ± 0.01 0.14 ± 0.01 – 1.79 ± 0.08 0.16 ± 0.02

– 0.19 ± 0.01 0.02 ± 0 2.78 ± 0.23 0.62 ± 0.04 0.31 ± 0.03 0.74 ± 0.03 2.72 ± 0.12 – 0.02 ± 0 0.02 ± 0 0.04 ± 0 0.04 ± 0 7.52 ± 0.32 0.04 ± 0 4.61 ± 0.41 0.1 ± 0.07 0.02 ± 0 0.08 ± 0 2.81 ± 0.21 0.02 ± 0 2.79 ± 0.20 0.02 ± 0 0.03 ± 0 0.03 ± 0 0.1 ± 0.01 0.26 ± 0 0.02 ± 0 0.04 ± 0 0.03 ± 0 0.14 ± 0.03 0.07 ± 0 0.02 ± 0 0.11 ± 0.03 0.1 ± 0.01 0.11 ± 0 0.02 ± 0 2.92 ± 0.26 0.05 ± 0 0.55 ± 0.06 0.21 ± 0.03 0.52 ± 0.04 0.34 ± 0.01 0.2 ± 0.02 0.1 ± 0.04 0.09 ± 0.02 0.13 ± 0 0.1 ± 0.01 0.08 ± 0.01 0.1 ± 0.01 – 0.15 ± 0.01 0.63 ± 0.04 1.42 ± 0.09 2.6 ± 0.17 2.5 ± 0.09 0.17 ± 0.01 0.05 ± 0.01 0.02 ± 0 1.86 ± 0.14 2.3 ± 0.22 0.51 ± 0.02 0.75 ± 0.03 0.27 ± 0.04 0.17 ± 0.02 12.58 ± 0.54 0.27 ± 0 0.03 ± 0 0.37 ± 0.03 0.47 ± 0 0.07 ± 0.01 0.08 ± 0.01 1.77 ± 0.19 0.14 ± 0.03

0.02 ± 0 0.57 ± 0.06 0.03 ± 0 7.42 ± 0.68 1.34 ± 0.11 0.68 ± 0.07 1.89 ± 0.07 7.16 ± 0.3 0.02 ± 0 0.04 ± 0 0.1 ± 0.02 0.06 ± 0 0.09 ± 0.02 7.36 ± 0.29 0.05 ± 0 5.27 ± 0.19 0.11 ± 0.06 0.02 ± 0 0.09 ± 0.02 3.87 ± 0.25 0.05 ± 0 2.94 ± 0.13 – 0.05 ± 0.01 0.04 ± 0 0.14 ± 0.02 0.26 ± 0.03 0.03 ± 0 0.04 ± 0 0.02 ± 0 0.11 ± 0 0.14 ± 0.06 – 0.07 ± 0.01 0.21 ± 0.02 0.1 ± 0.01 0.02 ± 0 3.49 ± 0.31 0.04 ± 0 0.49 ± 0.05 0.21 ± 0.04 0.2 ± 0.01 0.67 ± 0.05 0.99 ± 0.03 0.05 ± 0 0.12 ± 0.03 0.17 ± 0.02 0.14 ± 0.01 0.04 ± 0 0.16 ± 0.02 – 0.11 ± 0.03 0.77 ± 0.03 0.85 ± 0.08 3.29 ± 0.28 2.32 ± 0.1 0.15 ± 0 0.02 ± 0 0.05 ± 0 2.24 ± 0.2 3.23 ± 0.14 0.48 ± 0.02 0.88 ± 0.04 0.32 ± 0.02 0.17 ± 0.01 10.39 ± 0.42 0.19 ± 0.03 0.03 ± 0 0.29 ± 0.01 0.28 ± 0.02 0.09 ± 0.01 – 1.11 ± 0.07 0.24 ± 0

(continued on next page) 6

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Table 3 (continued) N°

compound

RI

RI (lit.)

HD

MAHD-SG

MAHD-CG

MASD-SG

MASD-CG

75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109

1-epi-cubenol γ- Eudesmol Hinesol α- Muurolol (=δ- Cadinol) Cubenol α- cadinol α- Eudesmol 7-epi-α-Eudesmol Longiborneol acetate Cadalene Eudesma-4(15),7-dien-1β-ol Acorenone Amorpha-4,9-diene-2-ol Eudesm-7(11)-en-4-ol 10-nor-Calamenen-10-one Mayurone iso-Longifolol Curcumenol 7, 14-anhydro-Amorpha-4,9-diene Cyclocolorenone Aristolone 14-hydroxy-α-Muurolene (E)-Isovalencenol 14-hydroxy-δ-Cadinene iso-LongifoloIacetate Cryptomeridiol β-Vetivone Eudesm-7(11)-en-4-ol, acetate (2E,6E)-Farnesyl acetate α-Chenopodiol Cubitene Totarene Carissone Z,E-Geranyl linalool Manool

1628 1633 1642 1646 1647 1655 1654 1664 1678 1685 1689 1694 1701 1703 1705 1712 1730 1735 1754 1760 1764 1782 1791 1806 1821 1815 1824 1841 1847 1857 1878 1925 1928 2001 2058

1627 1630 1640 1644 1645 1652 1652 1662 1684 1675 1687 1692 1700 1700 1702 1709 1728 1733 1755 1759 1762 1779 1793 1803 1819 1813 1822 1839 1845 1855 1878 1922 1926 1998 2056

0.3 ± 0.02 1.97 ± 0.08 6.1 ± 0.2 6.55 ± 0.33 1.66 ± 0.11 2.66 ± 0 17.89 ± 0.79 1.2 ± 0.09 1.21 ± 0.07 0.13 ± 0.02 0.07 ± 0.01 0.11 ± 0.02 0.08 ± 0.01 2.32 ± 0.19 0.14 ± 0.02 0.59 ± 0.04 0.15 ± 0.04 0.1 ± 0.03 0.07 ± 0 0.06 ± 0 0.04 ± 0 0.1 ± 0.01 0.1 ± 0.02 0.02 ± 0 0.08 ± 0.01 0.08 ± 0.02 0.35 ± 0.09 0.02 ± 0 0.05 ± 0 0.04 ± 0 0.03 ± 0 0.03 ± 0 0.03 ± 0 0.02 ± 0 0.02 ± 0

0.21 ± 0.01 1.24 ± 0.01 5.45 ± 0.17 4.76 ± 0.24 2.35 ± 0.08 1.59 ± 0.09 14.18 ± 0.36 0.68 ± 0.05 0.82 ± 0 0.07 ± 0.01 0.03 ± 0 0.08 ± 0 0.04 ± 0 1.14 ± 0.08 0.11 ± 0 0.38 ± 0.04 0.07 ± 0 0.05 ± 0 0.04 ± 0 0.03 ± 0 – 0.02 ± 0 0.04 ± 0 – 0.06 ± 0 – 0.14 ± 0.02 0.02 ± 0 0.02 ± 0 0.05 ± 0 0.02 ± 0 0.02 ± 0 – – –

0.16 ± 0.05 2.19 ± 0.14 4.85 ± 0.22 5.93 ± 0.41 2.33 ± 0.09 1.25 ± 0.11 15.54 ± 0.41 0.64 ± 0.01 1.25 ± 0.09 0.07 ± 0 0.04 ± 0 0.07 ± 0 0.05 ± 0 2.34 ± 0.24 0.14 ± 0.01 0.42 ± 0.02 0.11 ± 0.03 0.06 ± 0 0.05 ± 0 0.04 ± 0 – 0.05 ± 0 0.05 ± 0 – 0.07 ± 0.01 – 0.19 ± 0.04 0.02 ± 0 0.02 ± 0 0.04 ± 0 0.02 ± 0 0.02 ± 0 – – –

0.17 ± 0.04 2.44 ± 0.16 5.21 ± 0.19 5.64 ± 0.27 1.13 ± 0.12 1.91 ± 0.07 15.92 ± 0.75 0.61 ± 0.03 1.49 ± 0.14 0.06 ± 0.01 0.03 ± 0 0.06 ± 0 0.05 ± 0 2.45 ± 0.3 0.12 ± 0.01 0.32 ± 0.01 0.09 ± 0.01 0.06 ± 0 0.04 ± 0 0.04 ± 0 – 0.04 ± 0 0.06 ± 0 – – 0.07 ± 0 0.17 ± 0.02 0.05 ± 0 0.04 ± 0 0.07 ± 0.01 0.03 ± 0 0.03 ± 0 – – –

0.77 ± 0.06 0.1 ± 0.04 3.72 ± 0.31 3.77 ± 0.36 1.15 ± 0.07 1.56 ± 0.1 11.64 ± 0.5 0.62 ± 0.02 0.4 ± 0.08 0.06 ± 0 – 0.04 ± 0 0.02 ± 0 0.61 ± 0.09 0.04 ± 0 0.15 ± 0 0.03 ± 0 0.02 ± 0 0.02 ± 0 0.02 ± 0 – 0.02 ± 0 0.03 ± 0 – 0.03 ± 0 – 0.07 ± 0 0.02 0 0.03 ± 0 0.05 ± 0 0.02 ± 0 – – – –

14.91 8.47 25.40 50.37 0.18 99.33

20.2 13.62 26.19 39.28 0.08 99.37

20.86 12.52 23.49 42.79 0.07 99.73

14.96 11.02 29.60 43.88 0.1 99.56

26.66 13.06 29.63 30.28 0.06 99.69

Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Other components Total

RI (lit): retention indices of reference (Adams, 2007). RI: calculated retention indices. Values represent the average of three values; Mean ± SD (n = 3).

MAHD-GC essential oils, displaying a relative similarities in chemical classes percentages, whereas the third HD and MASD-SG regroups EOs marked the highest content in oxygenated sesquiterpenes. Surprisingly, cryogenic grinding caused a significative variability in qualitative and quantitative composition for MASD EOs, hence a non significative effect was noted for the MAHD EOs. From where, results suggested that extraction time (low for MASD compared to MAHD) and

significative variability between the EOs extracted by highlighted methods. The first three PCA axes explained 90.24% of information, and the representation was made in the two first axes (69.27% of information) (Fig. 3), PCA confirmed K-Means results revealing three separate groups. The group formed by MASD-CG essential oil has been previously characterized from other oils by it richness in monoterpene hydrocarbons (Table 3), the second group is formed by MAHD-SG and

Fig. 2. percentage of major constituents of Cymbopogon schoenanthus L. Spreng essential oil extracted by different techniques. 7

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varying according the method used (except for isopulegol (Fig. 5b) and cis-piperitol (Fig. 6a)). In general, for low molecular weight compounds like cis-piperitol (Fig. 6a), allo-ocimene (Fig. 4c) and isopulegol (Fig. 5b) we observed an increase in percentages to reach a maximum value in the first step of extraction as cis-piperitol (HD: 4.54% at 10 min; MAHD-SG: 6.41% at 2 min; MAHD-CG: 6.42% at 2 min; MASD-SG: 6.36% at 5 min and MASD-CG: 6.80% at 5 min); allo-ocimene (HD: 15.50% at 10 min; MAHD-SG: 19.66% at 2 min; MAHD-CG: 16.03% at 2 min; MASD-SG: 15.48% at 5 min and MASD-CG: 13.56% at 5 min) and isopulegol (HD: 11.37% at 10 min; MAHD-SG: 11.99% at 2 min; MAHD-CG: 10.05% at 2 min; MASD-SG: 9.96% at 5 min and MASD-CG: 8.52% at 5 min). This step is followed by a decreasing curvilinear line (second step) to reach the end of extraction (last step); this decreasing step is probably due simultaneously to the decrease of the relative percentage and the absolute value during the extraction time. This phenomenon can be attributed to many factors such as the low molecular weight of monoterpenes as well as the content of these components in exogenous and/ or endogenous sites. Other previous studies are in agreement with our findings (Benyoussef et al., 2005; Fornari et al., 2012; Tigrine-Kordjani et al., 2012). Whilst, for the compounds with high molecular weight such as transdauca-4(11),7-diene, Hinesol and α-muurolol (sesquiterpene hydrocarbons and oxygenated sesquiterpens), the yield increased during extraction time. Conversely, δ-2-carene, sylvestrene and β-elemene showed a decreasing profile for microwave techniques compared to HD, their percentage increased significatively during the first minutes of extraction then tending to a horizontal line indicating the end of the extraction process. Chemically, and as indicated in Figs. 4, 5 and 6, the essential oils collected in each time by MAHD-CG and MASD-CG were different to those obtained by MAHD-SG and MASD-SG respectively, this variation is probably caused by change in the structure inside the grinded plant in contact with water or water vapors during the extraction process. Indeed, physical structure of the plant affects the diffusion of oil from this matrix. Globally qualitative kinetic study proved that microwave extraction processes (internal heating) were fast compared to conventional hydrodistillation (external heating).Yet, the oxygenated compounds isolated in less amounts with microwave methods specially MASD-CG compared to long time extraction technique as HD do not signify a lower quality of microwave volatiles. Moreover, some of the oxygenated compounds (HD oils) can be degradation products due to thermal

Fig. 3. PCA for CS essential oils extracted by the five methods according to chemical composition.

the contact solid-liquid (no contact plant material-boiling water) can be responsible of this distinguished essential oils. 3.6. Effect of time (kinetic extraction) on chemical composition of Cymbopogon schoenanthus L. Spreng essential oil isolated by different methods In order to spot the influence of extraction time on chemical composition and behavior of CS L. Spreng essential oil during the extraction process, each volatile oil sample collected corresponding to each point of the curves presented in Fig. 1, was injected and analyzed by GC-FID and GC–MS analysis in the same conditions as the previous essential oils extracted. The accumulated percentages of ten major constituents of essential oils extracted by HD, MAHD-SG, MAHD-CG, MASD-SG and MASD-CG according to different times are presented in Figs. 4, 5 and 6. Regarding Fig. 4a α-eudesmol was present during all time of extraction varying according extraction technique (HD: 8.68–15.91%; MAHD-SG: 4.26–18.13%; MAHD-CG: 4.65–13.14%; MASD-SG: 4.31–12.21% and MASD-CG: 2.84–8.45%), showing a continuous increasing of this compound during the extraction process in the five highlighted methods especially in the first step of extraction (AB). Other major constituents such as trans-dauca-4(11),7-diene (Fig. 4b), alloocimene (Fig. 4c), α-muurolol (Fig. 4d), Hinesol (Fig. 5a), δ-2-carene (Fig. 5c), sylvestrene (Fig. 5d)and β-elemene (Fig. 6b), all of them were also present in high yield during the first time of extraction process,

Fig. 4. Accumulated percentages of major constituents according to extraction time: (a) α-eudesmol, (b) trans-dauca-4(11),7-diene, (c) allo-ocimene, (d) α-muurolol. 8

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Fig. 5. Accumulated percentages of major constituents according to extraction time: (a) Hinesol, (b) Isopulegol, (c) 2-δ-carene, (d) sylvestrene.

(08.67 ± 0.58 mm) and Candida albicans (08.67 ± 0.58 mm). In addition, essential oil isolated from MAHD-CG exhibited a high activity against Fusarium culmorum (27.00 ± 1.00 mm), moderate activity against Listeria monocytogenes (17.33 ± 0.29 mm) and Aspergillus westerdijkiae (15.33 ± 0.29 mm), weak to moderate activity against Aspergillus flavus (12.50 ± 0.29 mm), Staphylococcus aureus (11.50 ± 0.50 mm) and Candida albicans (10.00 ± 0.00 mm) and weak activity against Salmonella Typhi (08.00 ± 0.50 mm) and Klebsiella pneumoniae (07.67 ± 0.29 mm). For MASD-SG essential oil, results revealed a great activity against Fusarium culmorum (45.67 ± 1.15 mm), Listeria monocytogenes (26.17 ± 0.29 mm) and Aspergillus westerdijkiae (20.67 ± 1.15 mm), moderate activity against Aspergillus flavus (15.33 ± 0.58 mm) and weak to moderate activity against Staphylococcus aureus (14.50 ± 0.50 mm), Klebsiella pneumoniae (14.17 ± 0.29 mm), Candida albicans (10.67 ± 0.58 mm) and Salmonella Typhi (10.33 ± 0.29 mm). Finally, essential oil extracted by MASD-CG presented great activity against Fusarium culmorum (25.83 ± 0.29 mm) and Listeria monocytogenes (20.67 ± 0.29 mm), weak to moderate activity against Aspergillus flavus (11.17 ± 0.76 mm), Staphylococcus aureus (10.33 ± 0.29 mm), Aspergillus westerdijkiae (9.33 ± 0.58 mm) and Klebsiella pneumoniae (09.17 ± 0.29 mm) and weak activity against Salmonella Typhi (08.17 ± 0.29 mm) and Candida albicans (07.33 ± 0.58 mm). Table 4 indicates clearly that filamentous fungus Fusarium culmorum as well Listeria monocytogenes bacteria were strongly inhibited by C. schoenanthus L. Spreng essential oils specially MASD oil with classical

reactions. In addition, the essential oils present in the plants can contain more monoterpene and sesquiterpene hydrocarbons than those obtained during the heating process (Akloul et al., 2014; Benkaci-Ali et al., 2006). 3.7. Effect of extraction technique on antimicrobial activity The antimicrobial activity of Cymbopogon schoenanthus L. Spreng essential oils against tested microorganisms was qualitatively and quantitatively estimated by inhibition zone diameter and MIC values respectively. 3.7.1. Agar disc diffusion test Results show that inhibitory effect was detected on eight pathogens (Table 4). Essential oil extracted by HD showed a high activity against Fusarium culmorum (26.33 ± 0.58 mm) and Listeria monocytogenes (21.67 ± 0.58 mm), moderate activity against Staphylococcus aureus (15.33 ± 0.58 mm), weak to moderate activity against Klebsiella pneumoniae (11.67 ± 0.29 mm), Aspergillus flavus (10.33 ± 0.58 mm) and Salmonella Typhi (09.00 ± 0.50 mm) and week activity against Candida albicans (07.33 ± 0.58 mm) and Aspergillus westerdijkiae (06.50 ± 0.50 mm). Whereas, Essential oil extracted by MAHD-SG presented a high activity against Fusarium culmorum (20.33 ± 0.58 mm), moderate activity against Listeria monocytogenes (19.00 ± 1.00 mm), weak to moderate activity against Staphylococcus aureus (13.17 ± 0.29 mm), Aspergillus flavus (13.17 ± 0.29 mm), Klebsiella pneumoniae (10.50 ± 0.50 mm) and weak activity against Salmonella Typhi (08.67 ± 1.15 mm), Aspergillus westerdijkiae

Fig. 6. Accumulated percentages of major constituents according to extraction time: (a) cis-Piperitol, (b) β-Elemene. 9

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Table 4 Disk diffusion (mm) of Cymbopogon schoenanthus L. Spreng essential oils (10 μl) isolated by the five extraction methods. Microorganisms

Inhibition zone (mm) HD

Bacterial strains Staphylococcus aureus Listeria monocytogenes Klebsiella pneumoniae Salmonella Typhi Escherichia coli Fungal strains Aspergillus westerdijkiae Fusarium culmorum Aspergillus flavus Yeast Candida albicans

15.33 21.67 11.67 09.00 nd

± ± ± ±

0.58 0.58 0.29 0.50

MAHD-SG

MAHD-CG

13.17 19.00 10.50 08.67 nd

11.50 17.33 07.67 08.00 nd

± ± ± ±

0.29 1.00 0.50 1.15

± ± ± ±

MASD-SG

0.50 0.29 0.29 0.50

14.50 26.17 14.17 10.33 nd

± ± ± ±

0.50 0.29 0.29 0.29

MASD-CG

Amoxicillin (30 μg/disc)

10.33 20.67 09.17 08.17 nd

17.83 30.33 40.17 19.50 29.50

± ± ± ±

0.29 0.58 0.29 0.29

06.50 ± 0.50 26.33 ± 0.58 10.33 ± 0.58

08.67 ± 0.58 20.33 ± 1.15 13.17 ± 0.29

15.33 ± 0.29 27.00 ± 1.00 12.50 ± 0.50

20.67 ± 1.15 45.67 ± 1.15 15.33 ± 0.58

09.33 ± 0.58 25.83 ± 0.29 11.17 ± 0.76

nd nd nd

7.33 ± 0.58

8.67 ± 0.58

10.00 ± 0.00

10.67 ± 0.58

7.33 ± 0.58

nd

± ± ± ± ±

0.76 1.04 0.29 0.58 0.50

Aspergillus westerdijkiae (10–100 μg/ml) and Candida albicans (20–100 μg/ml). However, Escherichia coli and Salmonella Typhi were the most resistant bacteria against the oils tested. A variability of MIC values were noted according the technique achieved where HD, MAHDSG and MASD-CG essential oil was more efficient against Staphylococcus aureus, Candida albicans, Klebsiella pneumonia respectively. Nevertheless, MASD-SG EO was the most effective against the majority of tested microorganisms. This is may be due to the richness in some sesquiterpenes hydrocarbons (Trans- dauca-4(11),7-diene) and oxygenated (eudesmanes) previously mentioned. In fact, antimicrobial activity is highly dependent on the composition, thus, the suggestion of Bouchra et al. (2003) that oxygenated monoterpenes are particularly active against microbial cells, are in agreement with our results (Table 3) indicating a significant amount of oxygenated monoterpenes (8.47%–13.62%) consisted essentially of isopulegol (3.67–5.58%), cis-piperitol (2.07–3.87%) and trans-piperitol (1.97–3.13%). On the other hand, the presence of p-cymene (0.74–1.89%) can be responsible for the activity of CS essential oil (Oussalah et al., 2006). Although these compounds are considered to be minor components, they might contribute to an increase in activity (Lopes-Lutz et al., 2008; Mourey and Canillac, 2002; Romagnoli et al., 2005) in addition to the synergetic and antagonistic effects that must be taken in consideration (Saka et al., 2017). In order to highlight the variability of antimicrobial activity of Cymbopogon schoenanthus L. Spreng’s essential oils according to extraction technique, data analysis with K-means clustering and Principal Component Analysis (PCA) was also performed using R programming language, based on a scaled and centered matrix linking inhibition zones to extraction techniques (HD, MAHD-SG, MAHD-CG, MASD-SG and MASD-CG). Clustering classification permitted to distinguish two separate classes of essential oils; the class 1 including HD, MAHD-SG, MAHD-CG and MASD-CG essential oils and class 2 corresponding to MASD-SG

grinding probably due to it specific composition (Table 3). However, Escherichia coli was the most resistant strain where no inhibition zone was observed may be because the qualitative composition of the volatiles not containing specific compounds against this strain. Results also showed that Gram-positive bacteria were more sensitive to the action of investigated essential oils than Gram-negative bacteria. The intricacy of EOs mechanism of action on microorganisms generally depends on their hydrophilic or lipophilic character, the structure of microorganism’s cell as well as the arrangement of it external membrane (Kalemba and Kunicka, 2003). Generally, Gram-positive organisms are more week to the action of antibacterials (Vaara, 1992) due to the hydrophilic character of their exterior membrane (Nikaido, 1996) Antibacterials inhibit bacteria by destroying their cell walls in addition to their cytoplasmic membrane, causing the coagulation and leakage of microorganism’s cytoplasm. Researchers (Kalemba and Kunicka, 2003) affirmed that the action EOs on bacterial and fungal cells is related to the reticence of DNA, RNA, proteins and polysaccharides synthesis. In comparison with previous research (Hashim et al., 2016), it was mentioned that essential oil from Cymbopogon schoenanthus L. Spreng exhibited antibacterial activity against Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae. The effectiveness against Escherichia coli is probably be due to composition of essential oil dependent on the soil, climate, environmental and geographic conditions, harvest time and extraction method. 3.7.2. Determination of minimum inhibitory concentration (MIC) From the Table 5 corresponding to minimum inhibitory concentrations (MIC) values of each EO and in agreement with disc diffusion results, CS essential oils displayed a great efficacy against Fusarium culmorum (0.25–2 μg/ml) and Listeria monocytogenes (1–20 μg/ml), and moderate inhibition against Staphylococcus aureus (1–30 μg/ml), Klebsiella pneumoniae (50–60 μg/ml), Aspergillus flavus (10–50 μg/ml),

Table 5 Minimum inhibitory concentration (μg/ml) of Cymbopogon Schoenanthus L. Spreng volatile oils extracted by different methods. Microorganisms Bacterial strains Staphylococcus aureus Listeria monocytogenes Klebsiella pneumoniae Salmonella Typhi Escherichia coli Fungal strains Aspergillus westerdijkiae Fusarium culmorum Aspergillus flavus Yeast Candida albicans

HD

MAHD-SG

MAHD-CG

MASD-SG

MASD-CG

Amoxicillin (μg/ml)

1 10 20 > 150 > 150

5 2 50 > 150 > 150

30 20 60 > 150 > 150

5 1 5 100 > 150

2 2 30 > 150 > 150

1.25 0.25 0.1 0.5 0.25

100 1 50

30 2 30

30 0,5 30

10 0,25 50

30 1 10

nd nd nd

100

20

30

60

100

nd

10

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Fig. 7. PCA for antimicrobial activity of Cymbopogon schoenanthus L. Spreng essential oil based on matrix, linking the extraction methods to inhibition zones.

essential oil. Indeed, EO isolated by MASD-SG has shown a particularity in antimicrobial activity to be most effective essential oil on tested microorganisms. PCA (Fig. 7) confirmed K-Means results showing the two separate groups. Globally, our data about antimicrobial activity evaluated by disk diffusion method and determination of minimum inhibitory concentration revealed that essential oils from Cymbopogon schoenanthus L. Spreng extracted by the studied methods presented different behavior against tested microorganisms, noting that each strain was particularly more susceptible to one essential oil as to others.

4. Conclusion The microwave procedures (MAHD and MASD) offer important advantages over traditional hydrodistillation, namely: shorter extraction times, substantial savings of energy, and a reduced environmental burden. All these advantages make MAHD and MASD a good alternative for the extraction of essential oils from aromatic plants preserving their quality (refractive index and specific gravity). Cryogenic grinding (N2at −196 °C) allowed to increase yield MAHD-CG (1.76%) and MASD-CG (1.5%) compared to MAHD-SG (1.25%) and MASD-SG (1.11%), because the reduction of particle size and increasing surface for better solvent permeation. It has also preserved volatile compounds by eliminating their loss during classic grinding. Qualitatively, differences in composition according the technique used were confirmed. HD volatiles showed a predominance of oxygenated sesquiterpens while MASD-GC oil was rich of monoterpene hydrocarbons as confirmed by data analysis where K-means clustering and PCA permitted to distinguish between three separate classes of essential oils isolated from the different methods. The kinetic study showed that the qualitative and quantitative profile of EOs differed significatively according the technique and the grinding mode used. The cryogenic grinding affects the internal structure of the plant, hence, the mass transfer and the diffusion of essential oil toward the external medium allowing the acceleration of the extraction process as well as a qualitative and quantitative selective extraction of compounds from the exogenous and endogenous sides of the plant. This fluctuation in composition is directly correlated with antibacterial and antifungal properties closely related to the technique extraction and the kind of the grinding used. View the high activity of CS volatiles against Fusarium Culmorum, this plant can used to protect cereals or other seedlings threatened by this pathogen (Lawrence, 1978).

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